CN105176937A - Recombinant newcastle disease virus and application thereof in preparing anti-cancer drug - Google Patents

Abstract

The invention discloses a recombinant Newcastle disease virus and its application in preparing anticancer drugs. The preparation method of the recombinant Newcastle disease virus provided by the present invention comprises the following steps: co-transfecting mammalian cells with the recombinant plasmid pBrClone30/DR5-TRAIL, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid and culturing to obtain the obtained The recombinant Newcastle disease virus; the recombinant plasmid pBrClone30/DR5-TRAIL is a plasmid having a DNA molecule shown in nucleotides 2703-19872 from the 5' end of sequence 3 of the sequence table. The present invention also protects the application of the recombinant Newcastle disease virus in the preparation of the product; the function of the product is as follows (a) and/or (b) and/or (c): (a) inhibiting tumor cell proliferation; (b ) killing tumor cells; (c) treating tumors. The invention provides a brand-new method for improving TRAIL resistance to enhance anti-tumor effect, and has great application value for tumor treatment.

Description

A recombinant Newcastle disease virus and its application in the preparation of anticancer drugs

technical field

The invention relates to a recombinant Newcastle disease virus and its application in the preparation of anticancer drugs.

Background technique

Malignant tumor is one of the major diseases that endanger human health. At present, more than 11 million people suffer from cancer every year in the world, and more than 8 million people die of cancer.

For a long time, people have mainly used traditional surgical resection and radiotherapy and chemotherapy to treat cancer. These methods can control the development of tumors to a certain extent, but the curative effect on patients with advanced tumor spread is limited. Cells are severely traumatized.

Therefore, a new method for treating malignant tumors is urgently needed.

Contents of the invention

The object of the present invention is to provide a recombinant Newcastle disease virus and its application in the preparation of anticancer drugs.

The preparation method of the recombinant Newcastle disease virus provided by the invention (named rClone30/DR5-TRAIL virus) comprises the following steps: combining the recombinant plasmid pBrClone30/DR5-TRAIL, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid Transfect mammalian cells and culture to obtain the recombinant Newcastle disease virus; the recombinant plasmid pBrClone30/DR5-TRAIL is a DNA molecule having the sequence 3 of the sequence table from the 2703-19872 nucleotide at the 5' end plasmid. The DNA molecule shown in the sequence 3 of the sequence listing from the 2703-19872 nucleotide at the 5' end is the genomic DNA of the rClone30/DR5-TRAIL virus. The recombinant plasmid pBrClone30/DR5-TRAIL can specifically be the plasmid shown in sequence 3 of the sequence listing. The mammalian cells can specifically be BHK-21 cells.

The preparation method of rClone30/DR5-TRAIL virus is as follows:

(1) Co-transfect BHK-21 cells with the recombinant plasmids pBrClone30/DR5-TRAIL, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid (per 1×10 ⁶ cells transfected with 1 μg of recombinant plasmid ppBrClone30/ DR5-TRAIL, 0.5 μg pBL-N plasmid, 0.25 μg pBL-P plasmid and 0.1 μg pBL-L plasmid), placed in a 5% CO ₂ , 37°C environment for static culture for 72 hours;

(2) Take the transfected cells obtained in step (1), freeze and thaw repeatedly 3 times, collect the cell supernatant by centrifugation, then inoculate into the allantoic cavity of 9-11-day-old SPF chicken embryos, and culture at 37°C for 72 hours , collect chicken embryo allantoic fluid (which contains rClone30/DR5-TRAIL virus).

The preparation method of rClone30/DR5-TRAIL virus is as follows:

(1) Co-transfect BHK-21 cells with the recombinant plasmid pBrClone30/DR5-TRAIL, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid (per 1×10 ⁶ cells transfected with 1 μg of recombinant plasmid pBrClone30/ DR5-TRAIL, 0.5 μg pBL-N plasmid, 0.25 μg pBL-P plasmid and 0.1 μg pBL-L plasmid), placed in a 5% CO ₂ , 37°C environment for static culture for 72 hours;

(2) Take the transfected cells obtained in step (1), freeze and thaw repeatedly 3 times, collect the cell supernatant by centrifugation, then inoculate into the allantoic cavity of 9-11-day-old SPF chicken embryos, and culture at 37°C for 72 hours , collect chicken embryo allantoic fluid;

(3) Get the chicken embryo allantoic fluid obtained in step (2), inoculate it into the new 9-11 day-old SPF chicken embryo allantoic cavity, place it in a 37° C. environment and cultivate it for 72 hours, and collect the chicken embryo allantoic fluid;

(4) Get the chicken embryo allantoic fluid obtained in step (3), inoculate it into the new 9-11 day-old SPF chicken embryo allantoic cavity, place it in a 37° C. environment and cultivate it for 72 hours, and collect the chicken embryo allantoic fluid;

(5) Mix the chicken embryo allantoic fluid obtained in step (2), the chicken embryo allantoic fluid obtained in step (3) and the chicken embryo allantoic fluid obtained in step (4) to obtain a mixed solution, which is rClone30/DR5 -TRAIL virus liquid.

The present invention also protects a recombinant Newcastle disease virus (named rClone30/DR5-TRAIL virus), the corresponding DNA of its genome is shown in the 2703-19872 nucleotides from the 5' end of sequence 3 in the sequence listing.

The present invention also protects the application of the recombinant Newcastle disease virus described above in the preparation of products; the function of the product is as follows (a) and/or (b) and/or (c): (a) inhibiting tumor cells Proliferation; (b) killing tumor cells; (c) treating tumors. In the above (a) and/or the above (b), the tumor cells are liver cancer cells or glioblastoma cells. In the above (a) and/or the above (b), the tumor cells are human liver cancer cells or human glioblastoma cells. In said (a) and/or said (b), said tumor cells are HepG2 cells or U251 cells. In said (c), said tumor is liver cancer or glioma. In said (c), said tumor is a tumor caused by H22 cells.

The present invention also protects a product comprising the recombinant Newcastle disease virus described above; the function of the product is as follows (a) and/or (b) and/or (c): (a) inhibiting tumor cell proliferation; (b) killing tumor cells; (c) treating tumors. In the above (a) and/or the above (b), the tumor cells are liver cancer cells or glioblastoma cells. In the above (a) and/or the above (b), the tumor cells are human liver cancer cells or human glioblastoma cells. In said (a) and/or said (b), said tumor cells are HepG2 cells or U251 cells. In said (c), said tumor is liver cancer or glioma. In said (c), said tumor is a tumor caused by H22 cells.

Newcastle disease virus (Newacstlediseasevirus, NDV) is a non-segmented single-stranded negative-sense RNA virus belonging to the avian paramyxovirus genus of the Paramyxoviridae Paramyxoviridae subfamily. cells were not significantly affected. As a viral vector, NDV can drive the expression level of the gene carried by its own replication and amplification, and can also directly affect the apoptosis and necrosis of tumor host cells, thereby inhibiting the growth of tumor cells.

The present invention uses reverse genetic manipulation technology to simultaneously insert the DR5 gene segment and the TRAIL gene segment between the F gene and the HN gene of the Newcastle disease virus genome (the DR5 gene segment is the full-length gene of the human DR5 gene, which is localized in tumor cells after expression) surface; the TRAIL gene segment is the extracellular region of the human TRAIL gene, which is secreted and expressed outside the cell), and the recombinant virus rClone30/DR5-TRAIL was obtained. Newcastle disease virus itself can slightly up-regulate the expression of DR5 on the surface of tumor cells, and the inserted foreign gene DR5 can greatly up-regulate the expression of DR5, thereby improving the sensitivity of tumor cells to TRAIL. The joint expression of exogenous gene TRAIL on the genome of Newcastle disease virus reduces the re-administration of TRAIL and alleviates the pain caused by simultaneous injection of multiple drugs. The expressed TRAIL can further induce tumor cell apoptosis after combining with DR5. The invention provides a brand-new method for improving TRAIL resistance to enhance anti-tumor effect, and has great application value for tumor treatment.

Description of drawings

Figure 1 shows the expression of DR5 protein after the recombinant virus infected tumor cells.

Figure 2 shows the expression of TRAIL protein after the recombinant virus infected tumor cells.

Figure 3 is the relative expression level of DR5 gene.

Figure 4 is the relative expression level of TRAIL gene.

Figure 5 is the relative expression levels of caspase3 gene, caspase7 gene and caspase8 gene.

Fig. 6 is the inhibition rate of recombinant virus to HepG2 cells.

Figure 7 shows the inhibition rate of recombinant virus on U251 cells.

Figure 8 shows the killing effect of the recombinant virus on tumor cells when the infection dose is 1 MOI.

Figure 9 shows the killing effect of the recombinant virus on tumor cells when the infection dose is 10 MOI.

Fig. 10 is the tumor volume growth curve of 10 mice in the allantoic fluid group.

Figure 11 is the tumor volume growth curve of 10 mice in the rClone30 group.

Figure 12 is the tumor volume growth curve of 10 mice in the rClone30/IL2 group.

Figure 13 is the tumor volume growth curve of 10 mice in the rClone30/DR5 group.

Figure 14 is the tumor volume growth curve of 10 mice in the rClone30/TRAIL group.

Figure 15 is the tumor volume growth curve of 10 mice in the rClone30/DR5-TRAIL group.

Figure 16 shows the average volume of tumors in each test group.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged. Mouse anti-human DR5 monoclonal antibody: sigma, Cat. No. SAB4700538. FITC-labeled rabbit anti-mouse: santacruze, Cat. No. SC-358946. The PBS buffers used in the examples are all pH 7.2, 0.1M PBS buffers unless otherwise specified.

BHK-21 cells: ATCC, ATCC number is CRL-13001. HepG2 cells (human liver cancer cells): Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, catalog number TCHu72. U251 cells (human glioma cells): Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, catalog number TCHu58. Pancreatin: Sigma, catalog number 8049-47-6. H22 cells (mouse hepatoma cells): ATCC Company, the product catalog number is 58426. ICR mice: Changchun Yisi Experimental Animal Technology Co., Ltd.

Literature mentioning "pBrClone30 plasmid": "Enhancement of anti-tumor activity of Newcastledisease virus by the synergistic effect of cytosine deaminase", ZhengLV, AsianPacJCancer Ptev.; Harbin Boao Pharmaceutical Technology Development Co., Ltd.

Documents mentioning "pBL-N plasmid", "pBL-P plasmid" and "pBL-L plasmid": Genetically engineered Newcastlediseasevirus expressing interleukin2isapotential drug candidate for cancer immunotherapy, Fuliang Bai, Immunology letters.; Harbin Boao Pharmaceutical Technology Development Co., Ltd.

The pBrClone30 plasmid contains the NP gene, P gene, M gene, F gene, HN gene and L gene of Newcastle disease virus, wherein there are HpaI and MluI enzyme cutting recognition sites between the F gene and the HN gene. Mammalian cells were co-transfected with pBrClone30 plasmid, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid and cultured (pBL-N plasmid, pBL-P plasmid and pBL-L plasmid play auxiliary roles, and pBrClone30 plasmid provides virus whole genome) to obtain strain Clone30.

The specific method of the chicken hemagglutination (HA) test is as follows: (1) Add 50 μL of normal saline to each of the 1-12 wells in the first row of the hemagglutination plate; (2) Add 50 μL of the virus solution to be tested in the first well, mix After uniformity, suck 50 μL into the second well, and then dilute until the 10th well, and discard 50 μL after mixing well; (3) Add 50 μL of 1% chicken red blood cells to each of the 1-12 wells; (4) After shaking Stand at room temperature for 20-30min, and observe the result.

The specific method of the chicken hemagglutination inhibition (HI) test is as follows: (1) Add 25 μL of normal saline to each of the 1-12 wells of the hemagglutination plate; In the 2nd well, doubling dilute until the 10th well, and discard 25 μL after mixing in the 11th well; (3) Add 25 μL of the rClone30 virus solution (4 hemagglutination units) prepared in Example 1 to each of the 1-12 wells, Let stand at room temperature for 30 minutes; (4) add 25 μL of 1% chicken red blood cells to wells 1-12; (5) shake and let stand at room temperature for 30-40 minutes, and observe the results.

Example 1, Preparation of rClone30/DR5-TRAIL virus solution

1. Construction of recombinant plasmids

1. Synthesize the double-stranded DNA molecule shown in sequence 1 of the sequence listing.

In sequence 1 of the sequence listing, the 1st to 6th nucleotides from the 5' end are the recognition sequence of the restriction endonuclease HpaI, the 7th to 12th nucleotides are Kozak sequences, and the 13th to 1248th nucleotides are the recognition sequence of restriction endonuclease HpaI. The nucleotides are DR5 gene segment (full-length DR5 gene), and the 1249th to 1254th nucleotides are the recognition sequence of the restriction endonuclease MluI.

2. Digest the double-stranded DNA molecule obtained in step 1 with restriction endonucleases HpaI and MluI, and recover the digested product.

3. Digest the pBrClone30 plasmid with restriction endonucleases HpaI and MluI, and recover the vector backbone of about 18kb.

4. Ligate the digested product of step 2 with the vector backbone of step 3 to obtain the recombinant plasmid pBrClone30/DR5.

5. Synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing.

In Sequence 2 of the sequence listing, the 1st to 6th nucleotides from the 5' end are the recognition sequence of restriction endonuclease MluI, the 7th to 17th nucleotides are the Geneend sequence, and the 18th nucleotide It is an intergenic sequence (IG), the 19th to 29th nucleotides are the Genestart sequence, the 30th to 35th nucleotides are the Kozak sequence, and the 36th to 659th nucleotides are the TRAIL gene segment (the cell of the TRAIL gene Outer region), the 667th to 672nd nucleotides are the recognition sequence of the restriction endonuclease MluI.

6. Single-digest the double-stranded DNA molecule obtained in step 5 with the restriction endonuclease MluI, and recover the digested product.

7. Digest the recombinant plasmid pBrClone30/DR5 with the restriction endonuclease MluI, and recover the vector backbone of about 19kb.

8. Ligate the digested product of step 6 with the vector backbone of step 7 to obtain the recombinant plasmid pBrClone30/DR5-TRAIL. After sequencing, the nucleotide sequence of the recombinant plasmid pBrClone30/DR5-TRAIL is shown in sequence 3 of the sequence listing (in sequence 3 of the sequence listing, the 2703-19872 nucleotides from the 5' end are rClone30/DR5-TRAIL virus genome).

9. Digest the pBrClone30 plasmid with the restriction endonuclease MluI, and recover the vector backbone of about 18 kb.

10. Ligate the digested product of step 6 with the vector backbone of step 9 to obtain the recombinant plasmid pBrClone30/TRAIL.

11. Synthesize the double-stranded DNA molecule shown in sequence 4 of the sequence listing.

In sequence 4 of the sequence listing, the 1st to 6th nucleotides from the 5' end are the recognition sequence of the restriction endonuclease HpaI, the 7th to 12th nucleotides are Kozak sequences, and the 13th to 474th nucleotides are the recognition sequence of the restriction endonuclease HpaI. The nucleotides are the IL2 gene segment, and the 475th to 480th nucleotides are the recognition sequence of the restriction endonuclease MluI.

12. Double-digest the double-stranded DNA molecule obtained in step 11 with restriction endonucleases HpaI and MluI, and recover the digested product.

13. Digest the pBrClone30 plasmid with restriction endonucleases HpaI and MluI, and recover the vector backbone of about 18 kb.

14. Ligate the digested product of step 12 with the vector backbone of step 13 to obtain the recombinant plasmid pBrClone30/IL2.

2. Preparation of recombinant virus

1. Co-transfect BHK-21 cells with recombinant plasmids pBrClone30/DR5-TRAIL, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid (about 1 μg of recombinant plasmid pBrClone30/DR5-TRAIL per 1×10 ⁶ cells) TRAIL, 0.5 μg of pBL-N plasmid, 0.25 μg of pBL-P plasmid and 0.1 μg of pBL-L plasmid), placed in 5% CO ₂ , 37° C. for static culture for 72 hours.

2. Take the transfected cells obtained in step 1, freeze and thaw repeatedly 3 times, collect the cell supernatant by centrifugation, then inoculate into the allantoic cavity of 9-11-day-old SPF chicken embryos, culture at 37°C for 72 hours, and collect the chicken Embryo allantoic fluid.

3. Take the chicken embryo allantoic fluid obtained in step 2, inoculate it into the new 9-11 day old SPF chicken embryo allantoic cavity, place it in a 37° C. environment for 72 hours, and collect the chicken embryo allantoic fluid.

4. Take the chicken embryo allantoic fluid obtained in step 3, inoculate it into the new 9-11 day old SPF chicken embryo allantoic cavity, place it in a 37° C. environment for 72 hours, and collect the chicken embryo allantoic fluid.

5. Mix the chicken embryo allantoic fluid obtained in step 2, the chicken embryo allantoic fluid obtained in step 3, and the chicken embryo allantoic fluid obtained in step 4 to obtain a mixed solution. The HA titer of the mixture was 2 ¹¹ , and the HI titer was 2 ⁹ .

The mixed solution obtained in step 5 was named rClone30/DR5-TRAIL virus solution.

3. Preparation of control virus

The pBrClone30 plasmid was used to replace the recombinant plasmid pBrClone30/DR5-TRAIL to carry out steps 1 to 5 of step 2, and the obtained mixed solution was named rClone30 virus solution.

The recombinant plasmid pBrClone30/DR5 was used to replace the recombinant plasmid pBrClone30/DR5-TRAIL to carry out steps 1 to 5 of step 2, and the obtained mixed liquid was named rClone30/DR5 virus liquid.

The recombinant plasmid pBrClone30/TRAIL was used to replace the recombinant plasmid pBrClone30/DR5-TRAIL to carry out steps 1 to 5 of step 2, and the obtained mixed liquid was named rClone30/TRAIL virus liquid.

The recombinant plasmid pBrClone30/IL2 was used to replace the recombinant plasmid pBrClone30/DR5-TRAIL to carry out steps 1 to 5 of step 2, and the obtained mixed liquid was named rClone30/IL2 virus liquid.

Embodiment 2, chicken embryo stability detection of recombinant virus

The virus solution to be tested is the rClone30/DR5-TRAIL virus solution, rClone30/DR5 virus solution or rClone30/TRAIL virus solution prepared in Example 1.

Take 100 μL of the virus solution to be tested, dilute it with sterilized PBS buffer, and inoculate it into 9-11-day-old SPF chicken embryos through the allantoic cavity, and incubate at 37°C for 72 hours. The allantoic fluid of chicken embryos was collected, and the HA-positive allantoic fluid was used to inoculate 9-11-day-old SPF chicken embryos for continuous passage.

Take 100 μL each of the allantoic fluid of the first, third, fifth, eighth and 10th generation chicken embryos, and calculate the TCID ₅₀ per ml of virus fluid according to the Reed-Muench method.

The experiments were repeated three times, and the results were averaged.

The results are shown in Table 1. The results showed that rClone30/DR5-TRAIL virus solution, rClone30/DR5 virus solution and rClone30/TRAIL virus solution all had proliferation stability.

Table 1 HA titer and TCID ₅₀ of chicken embryo allantoic fluid in each generation

Example 3, detection of expression of DR5 protein after recombinant virus infected tumor cells

The virus solution to be tested is the rClone30/DR5-TRAIL virus solution, rClone30/DR5 virus solution, rClone30/TRAIL virus solution or rClone30 virus solution prepared in Example 1.

1. Take the HepG2 cells in the logarithmic growth phase, infect the virus solution to be tested at a dose of 1 MOI, and incubate at 37°C for 48 hours.

2. After completing step 1, take the cells, centrifuge at 1700r/min for 5min, collect the cell pellet, and wash twice with pre-cooled PBS buffer.

3. Resuspend the cell pellet obtained in step 2 with PBS buffer, add mouse anti-human DR5 monoclonal antibody (primary antibody) and incubate on ice for 40 minutes, then centrifuge at 1700r/min for 5 minutes, collect the pellet, and wash with pre-cooled PBS buffer Wash twice.

4. Resuspend the precipitate obtained in step 3 with PBS buffer, add FITC-labeled rabbit anti-mouse (secondary antibody) and incubate on ice in the dark for 40 minutes, then centrifuge at 1700r/min for 5 minutes to collect the precipitate.

5. Resuspend the precipitate obtained in step 4 with PBS buffer, and use flow cytometry for detection.

The results are shown in Figure 1. In Figure 1, A is the detection result of fluorescence intensity of HepG2 empty cells, B is the detection result of fluorescence intensity of HepG2 cells infected with rClone30 virus fluid, C is the detection result of fluorescence intensity of HepG2 cells infected with rClone30/TRAIL virus fluid, D is the detection result of fluorescence intensity of HepG2 cells infected with rClone30 The detection results of fluorescence intensity of HepG2 cells infected with rClone30/DR5-TRAIL virus liquid, and E is the detection result of fluorescence intensity of HepG2 cells infected with rClone30/DR5-TRAIL virus liquid. The results showed that: compared with HepG2 empty cells, infection with rClone30 virus solution, rClone30/TRAIL virus solution, rClone30/DR5 virus solution and rClone30/DR5-TRAIL virus solution could significantly up-regulate the expression level of DR5 protein on the surface of HepG2 cells; Compared with virus liquid, infection with rClone30/DR5 virus liquid and rClone30/DR5-TRAIL virus liquid can significantly up-regulate the expression level of DR5 protein on the surface of HepG2 protein.

Embodiment 4, detect the expression of TRAIL protein after recombinant virus infects tumor cell

The virus solution to be tested is the rClone30/DR5-TRAIL virus solution, rClone30/DR5 virus solution, rClone30/TRAIL virus solution or rClone30 virus solution prepared in Example 1.

1. Take the HepG2 cells in the logarithmic growth phase, infect the virus solution to be tested at a dose of 1 MOI, and incubate at 37°C for 48 hours.

2. After completing step 1, take the cell culture supernatant, and use the HumanTRAIL/TNFSF10 QuantikineELISAkit (DTRL00; R&D Company) to detect the level of TRAIL protein in the cell culture supernatant.

The results are shown in Figure 2. In Figure 2, A is the detection result of HepG2 empty cells, B is the detection result of HepG2 cells infected with rClone30 virus fluid; C: the detection result of HepG2 cells infected with rClone30/DR5 virus fluid; D: the detection result of HepG2 cells infected with rClone30/TRAIL virus fluid Detection results of HepG2 cells; E: detection results of HepG2 cells infected with rClone30/DR5-TRAIL virus solution. The results showed that, compared with HepG2 empty cells, infected rClone30 virus liquid and infected rClone30/DR5 virus liquid, infection with rClone30/TRAIL virus liquid and rClone30/DR5-TRAIL virus liquid could significantly up-regulate the expression level of TRAIL protein.

Example 5. Detection of mRNA expression changes of apoptosis-related genes.

The virus solution to be tested is the rClone30/DR5-TRAIL virus solution, rClone30/DR5 virus solution, rClone30/TRAIL virus solution or rClone30 virus solution prepared in Example 1.

1. Take the HepG2 cells in the logarithmic growth phase, infect the virus solution to be tested at a dose of 1 MOI, and incubate at 37°C for 48 hours.

2. After completing step 1, take cells, extract total RNA and reverse transcribe it into cDNA.

3. Using the cDNA obtained in step 2 as a template, real-time fluorescent quantitative PCR was used to detect the relative expression of DR5 gene, TRAIL gene, caspase3 gene, caspase7 gene and caspase8 gene (caspase3, caspase7 and caspase8 are apoptosis-related factors), and The β-actin gene was used as an internal reference gene.

The primer pair used to detect the DR5 gene is as follows:

Upstream primer: 5'-AGATTCTCCTGAGATGTGCCGGA-3';

Downstream primer: 5'-ACCACCTGAGCAGATGCCTTTCAGG-3'.

The primer pair used to detect the TRAIL gene is as follows:

Upstream primer: 5'-TCAGAGAGTAGCAGCTCACATA-3';

Downstream primer: 5'-GTTCACCATTCTCTCAAGTGCAA-3'.

The primer pair used to detect the caspase3 gene is as follows:

Upstream primer: 5'-TCATAAAAGCACTGGAATGACATC-3';

Downstream primer: 5'-TTCTGAATGTTTCCCCTGAGGTT-3'.

The primer pair used to detect the caspase7 gene is as follows:

Upstream primer: 5'-TCTATGTGCCCCGTCAGTA-3';

Downstream primer: 5'-ACATCCATACCTGTCGCTTT-3'.

The primer pair used to detect the caspase8 gene is as follows:

Upstream primer: 5'-CATTTGCATATTTAGCCGCCAAG-3';

Downstream primer: 5'-TTAAGAGTCCCAGGAATTCAGCAAC-3'.

The primer pair used to detect the β-actin gene is as follows:

Upstream primer: 5'-CGTGAAAAGATGACCCAGAT-3';

Downstream primer: 5'-ACCCTCATAGATGGGCACA-3'.

The relative expression level of DR5 gene is shown in Fig. 3 . The results showed that infection with rClone30/DR5 virus liquid and rClone30/DR5-TRAIL virus liquid increased the relative expression of DR5 gene, which was significantly different from infection with rClone30 virus liquid and infection with rClone30/TRAIL virus liquid. The results showed that the exogenous gene DR5 inserted into the Newcastle disease virus vector could be effectively expressed in tumor cells.

The relative expression of TRAIL gene is shown in Fig. 4 . The results showed that the expression of TRAIL gene was almost not expressed after infection with rClone30 virus liquid and rClone30/DR5 virus liquid; Compared with rClone30/DR5 virus solution, the difference is extremely significant. The results showed that the exogenous gene TRAIL inserted into the Newcastle disease virus vector could be effectively expressed in tumor cells.

The relative expression levels of caspase3 gene, caspase7 gene and caspase8 gene are shown in Figure 5. The results showed that the high expression of caspase3, caspase7 and caspase8 genes could be detected after infection with rClone30/DR5-TRAIL virus liquid, and the difference was extremely significant compared with infection with rClone30 virus liquid, infection with rClone30/DR5 virus liquid, and infection with rClone30/TRAIL virus liquid.

Embodiment 6, the effect of recombinant virus on tumor cells

The virus solution to be tested is the rClone30/DR5-TRAIL virus solution, rClone30/DR5 virus solution, rClone30/TRAIL virus solution or rClone30 virus solution prepared in Example 1. Tumor cells were HepG2 cells and U251 cells.

1. MTT assay to detect the inhibitory effect of rClone30/DR5-TRAIL virus on tumor cells

1. Trypsinize the tumor cells in the logarithmic growth phase, and then use DMEM medium containing 10% calf serum to make a cell suspension of 2×10 ⁴ cells/mL.

2. Inoculate the cell suspension obtained in step 1 into a 96-well plate (200 microliters per well), place it in a 5% CO ₂ , 37°C environment and culture it for 24 hours, discard the culture supernatant, and wash with PBS buffer .

3. After completing step 2, get the 96-well plate and infect the virus solution to be tested (the following infection doses are set respectively: 0.1MOI, 1MOI or 10MOI; 100 μL of virus solution per hole; set with an equal volume of 5% calf serum DMEM medium instead of the virus solution (control group), placed in 5% CO ₂ , 37°C environment for static culture for 1 hour, discarded the culture supernatant, and washed with PBS buffer.

4. After completing step 3, take the 96-well plate, add DMEM medium containing 10% calf serum to each well, and place it in a 5% CO ₂ , 37°C environment for static culture for 24h, 48h or 72h.

5. After completing step 4, take the 96-well plate, add 20 μL MTT solution (5 mg/mL) to each well and incubate at 37°C for 4 hours, discard the culture supernatant, add 150 μL DMSO to each well, shake and mix for 10 minutes, and use a microplate reader The OD value at 490nm was measured, and the inhibition rate of cell growth was calculated.

Inhibition rate = (OD value of control treated wells - OD value of test treated wells) / OD value of control treated wells.

The inhibition rate of HepG2 cells is shown in Figure 6. In Fig. 6, A corresponds to the result of infecting different MOI (0.1MOI, 1MOI, 10MOI) of the virus solution to be tested in step 3 after culturing for 72 hours, and B corresponds to the results of infecting the same MOI (ie 0.1MOI) of the virus solution to be tested in different cultures Time (24h, 48h, 72h respectively) results, C corresponds to the results of infection with the same MOI (i.e. 1MOI) at different incubation times (respectively 24h, 48h, 72h), D corresponds to the results of infection with the same MOI (i.e. 10MOI ) The results of the virus liquid to be tested at different incubation times (respectively 24h, 48h, 72h).

The inhibition rate on U251 cells is shown in Figure 7. In Fig. 7, A corresponds to the result of infecting different MOI (0.1MOI, 1MOI, 10MOI) of the virus solution to be tested in step 3 after culturing for 72 hours, and B corresponds to the results of infecting the same MOI (ie 0.1MOI) of the virus solution to be tested in different cultures Time (24h, 48h, 72h respectively) results, C corresponds to the results of infection with the same MOI (i.e. 1MOI) at different culture times (respectively 24h, 48h, 72h), D corresponds to the results of infection with the same MOI (i.e. 10MOI ) The results of the virus liquid to be tested at different incubation times (respectively 24h, 48h, 72h).

The results showed that the inhibitory effect of rClone30/DR5-TRAIL virus on tumor cells was significantly stronger than that of rClone30 virus, rClone30/DR5 virus and rClone30/TRAIL virus. Proportional.

2. AnnexinV/PI method to detect the killing effect of rClone30/DR5-TRAIL virus on tumor cells

1. Trypsinize the HepG2 cells in the logarithmic growth phase, and then use DMEM medium containing 10% calf serum to make a cell suspension of 1×10 ⁴ cells/mL.

2. Inoculate the cell suspension obtained in step 1 into a 6-well plate (2ml per well), place it in a 5% CO ₂ , 37°C environment and culture it statically for 24 hours, discard the culture supernatant, and wash with PBS buffer.

3. After completing step 2, get the 6-well plate and infect the virus solution to be tested (the following infection doses are set respectively: 1MOI or 10MOI; the blank control of replacing the virus solution with an equal volume of DMEM medium containing 5% calf serum is set. ), placed in an environment of 5% CO ₂ and 37° C. for static culture for 1 hour, discarded the culture supernatant, and washed with PBS buffer.

4. After completing step 3, take the 6-well plate, add DMEM medium containing 10% calf serum to each well, and place it in an environment of 5% CO ₂ and 37°C for static culture for 48 hours.

5. After completing step 4, get the 6-well plate, collect the cells, digest the cells with DMEM medium containing 0.25% (mass ratio) trypsin, then wash twice with PBS buffer, then take (0.5-1) For ×10 ⁶ cells, gently suspend the cells with 500 μL BindingBuffer, add 10 μL FITC-labeled Annexin-V, then add 5 μL PI, mix well, react in the dark for 5-15 minutes, and then quantitatively detect with flow cytometry.

The results when the infection dose was 1 MOI are shown in Figure 8. In Figure 8, A is the test result of the blank control, B is the test result of HepG2 cells infected with rClone30 virus solution, C is the test result of HepG2 cells infected with rClone30/DR5 virus solution, and D is the test result of HepG2 cells infected with rClone30/TRAIL virus solution The detection results of cells, E is the detection results of HepG2 cells infected with rClone30/DR5-TRAIL virus solution.

The results when the infection dose was 10 MOI are shown in Fig. 9 . In Figure 9, A is the test result of the blank control, B is the test result of HepG2 cells infected with rClone30 virus solution, C is the test result of HepG2 cells infected with rClone30/DR5 virus solution, and D is the test result of HepG2 cells infected with rClone30/TRAIL virus solution The detection results of cells, E is the detection results of HepG2 cells infected with rClone30/DR5-TRAIL virus solution.

The results showed that rClone30/DR5-TRAIL virus, rClone30 virus, rClone30/DR5 virus and rClone30/TRAIL virus could all induce tumor cell apoptosis, and the degree of cell apoptosis was dose-dependent on the virus dose, rClone30/DR5-TRAIL The effect of virus was significantly stronger than that of rClone30 virus, rClone30/DR5 virus and rClone30/TRAIL virus.

Example 7, Therapeutic Effect of Recombinant Virus on Tumor and Detection of Virus Safety

1. The therapeutic effect of rClone30/DR5-TRAIL virus on tumor

1. Establish H22 liver cancer animal model

(1) Take H22 cells and suspend them with PBS buffer to obtain a cell suspension of 10 ⁶ cells/mL.

(2) Get 6-week-old ICR mice, subcutaneously inject 0.2 mL of the cell suspension prepared in step (1) into each right groin, and raise the mice normally. After 10 days, the mice that form solid tumors with a diameter of 5-8 mm, is the model mouse.

2. The therapeutic effect of rClone30/DR5-TRAIL virus on tumor

The model mice were randomly divided into six groups, 10 in each group, and were treated as follows:

rClone30/DR5-TRAIL group: Inject 0.2 mL of rClone30/DR5-TRAIL prepared in Example 1 into the solid tumor of each model mouse on the first day, the third day, the fifth day and the seventh day of the experiment Virus liquid (containing 10 ⁷ pfu virus);

rClone30/DR5 group: on the first day, the third day, the fifth day and the seventh day of the experiment, inject 0.2 mL of the rClone30/DR5 virus liquid prepared in Example 1 into the solid tumor of each model mouse (containing 10 ⁷ pfu virus);

rClone30/TRAIL group: on the first day, the third day, the fifth day and the seventh day of the experiment, inject 0.2 mL of the rClone30/TRAIL virus solution prepared in Example 1 into the solid tumor of each model mouse (containing 10 ⁷ pfu virus);

rClone30/IL2 group: on the first day, the third day, the fifth day and the seventh day of the experiment, inject 0.2 mL of the rClone30/IL2 virus solution prepared in Example 1 into the solid tumor of each model mouse (containing 10 ⁷ pfu virus);

rClone30 group: on the first day, the third day, the fifth day and the seventh day of the experiment, inject 0.2mL of the rClone30 virus solution prepared in Example 1 (containing 10 ⁷ pfu virus );

Allantoic fluid group: On the first day, the third day, the fifth day and the seventh day of the experiment, inject 0.2mL chicken embryo allantoic fluid into the solid tumor of each model mouse.

The tumor volume was measured every two days, and the tumor volume growth curve was drawn.

From the 0th day of the test (the 0th day refers to before the injection of the virus liquid) to the 14th day of the test, the tumor volume growth curves of 10 mice in the allantoic fluid group are shown in FIG. 10 . From the 0th day of the experiment (the 0th day refers to before the injection of the virus solution) to the 14th day of the experiment, the tumor volume growth curves of 10 mice in the rClone30 group are shown in FIG. 11 . From the 0th day of the experiment (the 0th day refers to before the injection of the virus solution) to the 14th day of the experiment, the tumor volume growth curves of 10 mice in the rClone30/IL2 group are shown in FIG. 12 . From the 0th day of the experiment (the 0th day refers to before the injection of the virus solution) to the 14th day of the experiment, the tumor volume growth curves of 10 mice in the rClone30/DR5 group are shown in FIG. 13 . From the 0th day of the test (the 0th day refers to before the injection of the virus solution) to the 14th day of the test, the tumor volume growth curves of 10 mice in the rClone30/TRAIL group are shown in FIG. 14 . From the 0th day of the experiment (the 0th day refers to before the injection of the virus solution) to the 14th day of the experiment, the tumor volume growth curves of 10 mice in the rClone30/DR5-TRAIL group are shown in FIG. 15 . From the 0th day of the test (the 0th day refers to before the injection of the virus solution) to the 14th day of the test, the average tumor volume of each test group is shown in FIG. 16 .

The results showed that, compared with the PBS group, rClone30 virus solution, rClone30/IL2 virus solution, rClone30/DR5 virus solution, rClone30/TRAIL virus solution and rClone30/DR5-TRAIL virus solution had a very significant inhibitory effect on tumors, rClone30 virus solution , rClone30/IL2 virus solution, rClone30/DR5 virus solution, rClone30/TRAIL virus solution had no significant difference in effect, rClone30/DR5-TRAIL virus solution and Clone30 virus solution, rClone30/IL2 virus solution, rClone30/DR5 virus solution, rClone30 /TRAIL virus solution has a significant difference.

2. Safety detection of rClone30/DR5-TRAIL virus

1. Acute toxicity test

Ten healthy 4-6 week-old ICR mice, half male and half male, were intraperitoneally injected with rClone30/DR5-TRAIL virus liquid (containing 10 ⁷ pfu virus), and observed for 48 hours after injection.

None of the mice had any of the following adverse reactions: inhibited breathing, unsteady limbs, paralysis, convulsions, tremors, and death.

2. Subacute toxicity test

Ten healthy 4-6 week-old ICR mice, half male and half male, were intraperitoneally injected with rClone30/DR5-TRAIL virus solution (containing 10 ⁷ pfu virus), and observed for 4 weeks after injection.

The water intake, food intake, fur color, and body weight of the mice were all normal, without any adverse reactions or deaths.

The results showed that the rClone30/DR5-TRAIL virus had no adverse effect on the normal growth of mice and was safe and reliable.

Claims (10)

1. a recombinant Newcastle disease virus, its preparation method comprises the steps: recombinant plasmid pBrClone30/DR5-TRAIL, pBL-N plasmid, pBL-P plasmid and pBL-L plasmid co-transfection mammalian cell and cultivates, and obtains described recombinant Newcastle disease virus; Described recombinant plasmid pBrClone30/DR5-TRAIL is for having the plasmid of sequence 3 from the DNA molecular shown in 5 ' end 2703-19872 position Nucleotide of sequence table.

2. recombinant Newcastle disease virus as claimed in claim 1, is characterized in that: the plasmid shown in sequence 3 that described recombinant plasmid pBrClone30/DR5-TRAIL is sequence table.

3. recombinant Newcastle disease virus as claimed in claim 1 or 2, is characterized in that: described mammalian cell is BHK-21 cell.

4. a recombinant Newcastle disease virus, DNA corresponding to its genome is if the sequence 3 of sequence table is from shown in 5 ' end 2703-19872 position Nucleotide.

5. the arbitrary described application of recombinant Newcastle disease virus in preparing product in Claims 1-4; The function of described product is following (a) and/or (b) and/or (c): (a) inhibition tumor cell is bred; (b) killing tumor cell; (c) treatment tumour.

6. apply as claimed in claim 5, it is characterized in that: in described (a) and/or described (b), described tumour cell is liver cancer cell or neurogliocytoma cell.

7. apply as claimed in claim 5, it is characterized in that: in described (c), described tumour is liver cancer or neurogliocytoma.

8. a product, comprises arbitrary described recombinant Newcastle disease virus in Claims 1-4; The function of described product is following (a) and/or (b) and/or (c): (a) inhibition tumor cell is bred; (b) killing tumor cell; (c) treatment tumour.

9. product as claimed in claim 8, it is characterized in that: in described (a) and/or described (b), described tumour cell is liver cancer cell or neurogliocytoma cell.

10. product as claimed in claim 8, it is characterized in that: in described (c), described tumour is liver cancer or neurogliocytoma.